1. Field of the Invention
The application relates to the quantitation of the protein HMGB1 or of the antibodies specific for HMGB1 in biological sample, in particular serum and CerebroSpinal Fluid (CSF), and their respective correlation with prognostic methods of the state of progression of neurological disorders or toward neurological disorders, in particular neurological disorders associated with HIV infection and with diagnostic methods. The invention also relates to the correlation of the protein HMGB1 or of the antibodies specific for HMGB1 with the monitoring of HIV infection or with viral load as well as prognostic methods of the state of progression of AIDS.
2. Description of the Related Art
Shortly after infection, HIV-1 is able to penetrate the brain, eventually resulting in HIV-1 associated complications in the Central Nervous System (CNS). HIV associated dementia (HAD) is clinically characterized by motor and behavioral dysfunctions leading to seizures, coma, and death within 6 months of onset. HIV-encephalitis, the pathological correlate of HAD, is characterized by widespread astrogliosis, oxidative stress, cytokine/chemokine dysregulation, and neuronal degeneration (Gonzalez-Scarano and Martin-Garcia, Nat Rev Immunol 2005, 5: 69-81). Since neurons are not infected by HIV-1, the current thinking is that these cells are damaged indirectly by pro-inflammatory chemokines released by activated glial cells. IP-10 (CXCL10) is a neurotoxic chemokine that is upregulated in astroglia and has been suggested to enhance retrovirus infection and mediate neuronal injury. Elevation of IP-10 in CSF was reported in HIV-infection, and its level correlated with CSF HIV viral load (Cinque P et al. J Neuroimmunol 2005, 168:154). It was also reported a possible association between CSF macrophage chemoattractant protein 1 (MCP-1 or CCL2) levels and the development of HAD in a HAART-experienced cohort with advanced HIV infection (Sevigny J J, Albert S M, McDermott M P; et al. Neurology. 2004;63:2084). Another study reported an association between plasma MCP-1 levels and HIV-associated dementia (Sevigny et al. Arch. Neurol. 2007, 64:97).
The high mobility group box 1 (HMGB1) protein is a non-histone chromosomal protein that acts as a potent proinflammatory cytokine when actively secreted from activated macrophages, monocytes, dendritic cells or other cells such as NK cells. HMGB1 behaves as a trigger of inflammation, attracting inflammatory cells, and of tissue repair, recruiting stem cells and promoting their proliferation. Moreover, HMGB1 activates dendritic cells (DCs) and promotes their functional maturation and their response to lymph node chemokines. Activated leukocytes actively secrete HMGB1 in the microenvironment. Thus, HMGB1 acts in an autocrine/paracrine fashion and sustains long-term repair and defense programs (Bianchi and Manfredi, 2007; Lotze and Tracey, 2005).
In recent studies, HMGB1 was shown to trigger HIV-replication in HIV-infected DC, thus contributing to the constitution of viral reservoirs in DCs (Saidi H, Melki M-T, Gougeon M-L, PLoS One 2008). Considering that DCs are the first targets for HIV in the first hours of mucosal infection, which will then migrate to secondary lymphoid organs where they will transmit HIV to T cells, these findings challenge the question of the in vivo involvement of HMGB1 in the triggering of viral replication and replenishment of viral reservoirs. HMGB1 was produced during a cross-talk between HIV-infected DCs and activated NK cells, also resulting in resistance of HIV-infected DCs to NK killing. DC survival was associated with the upregulation of two apoptosis inhibitors, c-IAP2 and c-FLIP in infected DCs, a process induced by HMGB1 (Melki M-T et al. PLoS Pathogens 2010, 6 (4) e1000862). Blocking HMGB1 activity by specific inhibitors, such as glycyrrhizin or blocking antibodies, abrogates HIV replication in infected DCs (Saidi H, Melki M-T, Gougeon M-L, PLoS One 2008), and restores the susceptibility of infected DCs to NK killing (Melki M-T et al. PLoS Pathogens 2010, 6(4) e1000862). These findings, which provide new insights into how HIV hijacks DCs to promote viral dissemination and to maintain viability of long-term reservoirs, have made the object of patent application PCT/EP2009/06828.
These findings also challenge the question of the in vivo involvement of HMGB1 in the triggering of viral replication and replenishment of viral reservoirs. To address this question, HMGB1 concentration in sera from HIV-infected patients have been quantified (Elisa, Shino test, IBL) to assess the in vivo contribution of circulating HMGB1 to plasma HIV viral load and to disease evolution. Moreover, considering that auto-antibodies specific for HMGB1 could be found in autoimmune diseases such as SLE (lupus) (Hayashi et al., 2009), a specific Elisa assay was developed to check whether anti-HMGB1-specific antibodies in sera from HIV-infected patients were detected in HIV disease.
The measure of both HMGB1 and anti-HMGB1 antibodies in patients' sera has been reported in patent application PCT/EP2009/06828. The following conclusions have been drawn up:
(i) chronic HIV infection triggers the production of HMGB1, detected at increased levels in sera from infected patients, which in turn induces the production of neutralizing antibodies;
(ii) an inverse correlation is detected between HMGB1 and anti-HMGB1 antibodies (Abs), indicating that when HMGB1 is bound to the antibodies, it is not detected anymore in serum samples;
(iii) the more anti-HMGB1 antibodies (meaning the more HMGB1 produced earlier) in sera, the less CD4 T cells, suggesting that increased levels of serum anti-HMGB1 antibodies are associated with disease evolution; and
(iv) potent anti-retroviral therapy (HAART) reduces serum levels of both HMGB1 and anti-HMGB1 antibodies and may normalize them under baseline levels.
Patent application PCT/EP2009/06828 also made the hypothesis that the more anti-HMGB1 antibodies in sera, the less serum viral load. However, this hypothesis has not been confirmed in a thorough analysis of the experiments performed and in a greater cohort of patients. Indeed, the correlation established between anti-HMGB1 antibodies and serum viral load disclosed in patent PCT/EP2009/06828 was further shown to derived from an improper statistical analysis of the results derived from a cohort of patients encompassing both untreated patients and treated patients (treatment having an effect on the viral load).